Week 1 & 2
Stella, good but include your Nanodrop images. Dr. B 090913
Friday 9/6----------
Agarose Gel:
Ladder failed to show due to a punctured well, and PCR will need to be repeated.
Figure 1: Agarose gel results of PCR of pgbr22 plasmid showing distinct bands on wells 2-4, corresponding to samples A, B, and C, respectively, but with no ladder for comparison due to the first well being punctured.
Monday 9/2 -------------
Nanodrop Practice:
Objective was to determine the concentration of pgbr22 plasmid.
Graphic results located in lab book (will upload graphs 9/9)
The concentrations recorded were low on average, possibly due to contents settling in the tube. Due to the low average, I used Imran's average concentration for determining amount of plasmid to be used for DNA sequencing and PCR.
DNA Sequencing:
Sample of pgbr22 was submitted and returned, but then I received a notification that it was run again. Final results are:
Where N's are base-pairs unable to be read by the sequencing machine. Due to the large number of N's, and the fact that the first sequencing failed, the sample must have been contaminated in some way.
Week 1 & 2
Stella, good but include your Nanodrop images. Dr. B 090913
Friday 9/6----------
Agarose Gel:
Ladder failed to show due to a punctured well, and PCR will need to be repeated.
Figure 1: Agarose gel results of PCR of pgbr22 plasmid showing distinct bands on wells 2-4, corresponding to samples A, B, and C, respectively, but with no ladder for comparison due to the first well being punctured.
Monday 9/2 -------------
Nanodrop Practice:
Objective was to determine the concentration of pgbr22 plasmid.
Graphic results located in lab book (will upload graphs 9/9)
The concentrations recorded were low on average, possibly due to contents settling in the tube. Due to the low average, I used Imran's average concentration for determining amount of plasmid to be used for DNA sequencing and PCR.
DNA Sequencing:
Sample of pgbr22 was submitted and returned, but then I received a notification that it was run again. Final results are:
NNNNNNNNNNNNNNNNNNNNNCNNGNNNNGNNNNNNCCNTNNNNNCGNNNNNNNNNNNNNNGNGNNNCTCANNGNGGNNNNATGNNNNNNGCGANNNNNNGNGNNGAGNANAGNNANACCNCCNNNNTNNACGNTCGNGNNCNNANNNNNNNTNAANACTTCCANTNNNNNNNNGCCNANCNNCCGTTNCNCNANNNNTTTGGANGCNGNCTNNNTNTNNNNTNGNNATGCGNACTTGCNGNANAGNTGTNTCACTTTNCAANAATGNAGCCNTANACNNTTNNCCANGTNCCNTNNNNGANNCNNNCNNCNNNGCANCCCGTNNGGTTCCCTGCCNNGTGGAGGCTNNNGNNTNNNANGNNNANNNNNCNGAANNNTNNGNAACANATNTTGTCNCTNTANATGANCTCNNTTNNCNNGGATNGTGNNNNCNTTGNNNNNNNTACNCNANGCANANNCNTTACGTANNNNGCNTGACANGGNAGATNCNNNNNGGACTGACNGNTNNNNTAACCNNGGATGTCTNCNTGGTNAANTNNNNAAAGANNNNNCNNNNNNNNGGGANGGNGCNNNTNNANTNNNNNNNTCNNNNNNCNGANGTNNNNNNNNNNGNCNNCTGTNGNNACNNANTGTGNNNNNNTNNNCNGNNNN
Where N's are base-pairs unable to be read by the sequencing machine. Due to the large number of N's, and the fact that the first sequencing failed, the sample must have been contaminated in some way.
Oligo Design:
Primers for phosphoatase 2C from T. gondii:
30 Oligos:
1 ATGGCGGACGCTAAGACCCTGCTGGGCAAGGTTAAACGTGCGACGGGTATGGGTG 55
2 GCGGTGTATTTCGGTTTTTTTGCTACAGAAGGACCTTCACCAACACCCATACCCGTCGCA 60
3 CAAAAAAACCGAAATACACCGCGACCGTTCCTGGCTTCACCCCGCCATCCGGTGATCAGC 60
4 ATAAACTCGCCGGACGTGTCAACAGCCATAAATTCGTTCATACGCTGATCACCGGATGGC 60
5 CACGTCCGGCGAGTTTATGCGCCACCTCTACATCGAAGAGGGCCGTACCGTGTGTGCGTC 60
6 GTGCGGAGACTCAGAGGTTGGACGACGATTGCGAGAGGTCGCAGACGCACACACGGTACG 60
7 ACCTCTGAGTCTCCGCACTCTGATGACGTTGTTGTGGTAGAAGGCATGCTCCGTGGCCGT 60
8 CGACCTTGGAAACCGTCGAACATAGCGTGAACACGGGTTTCCGGACGGCCACGGAGCATG 60
9 CGACGGTTTCCAAGGTCGCCATTCTGCTATGTGGCTGGCCCAGAACGTAATGAACTACCT 60
10 GGGTGATTTCTTCCTCGTTCACGTCGCGGAGATCGTTCAGGTAGTTCATTACGTTCTGGG 60
11 TGAACGAGGAAGAAATCACCCGTCAGTTCGAGCGCATGGACGGTGACCTCCGTGCAGCAA 60
12 TAACGAACGAAAATGATGAGCGCAGAGCTGCCGCCAGGCAGGTTTGCTGCACGGAGGTCA 60
13 CGCTCATCATTTTCGTTCGTTACGAGAAAAAGCCGACCGAAGCGCGTGTAGTTGGTCGTC 60
14 CGGCCACAGAGGTGAATTCCTTCGCACCCTCCGGAACGATTTGACGACCAACTACACGCG 60
15 GAATTCACCTCTGTGGCCGAGGCACTCGGTGGTCCGCTGATGCCGGTTGTTGCAATGAAT 60
16 ATGGTGTAAATACCTTTCGCAGCGCGAGGGTCACGACGGAAATTCATTGCAACAACCGGC 60
17 CTGCGAAAGGTATTTACACCATTCATGTTGCGTCTCTGGGTAACTCTCGTTGCGTTCTGA 60
18 TATGCGGGGTAGACAGGTGGATCGCAGTGCGACCAGATTTCAGAACGCAACGAGAGTTAC 60
19 CACCTGTCTACCCCGCATACCGCGTCTTCTCACAAAGAACGTCACCGCGTTCAAGCAGCG 60
20 CCGCCGAGGAGGAGTTCGCCGTTAACAGTGGTAAACACACCACCCGCTGCTTGAACGCGG 60
21 GAACTCCTCCTCGGCGGTGTTGTACCGATGACCCGTGCGTTCGGCTCTTTCGACTTCAAA 60
22 AGAAACCAGGTCTTGTTGGAGCTTACCTTGGCCACCTTTTTTGAAGTCGAAAGAGCCGAA 60
23 CTCCAACAAGACCTGGTTTCTGCGGTTCCAGACGTTACCACCTTCTTTGCGTATCCTGGT 60
24 AGTGCGCAAAGGCACCCGCGGTACCTGCTACGATGTCGTCACCAGGATACGCAAAGAAGG 60
25 GGTGCCTTTGCGCACTTCCGCTCTCATGCCGCGATTGCAGCCGCTATTGCGCTGTACCCG 60
26 TACCATGGCCTTGGCGGCGTCCAGAACCGTTTCAGGAGATACCGGGTACAGCGCAATAGC 60
27 CCGCCAAGGCCATGGTAGTCAATGCCAAACGTCGTAAAGTTACCAAAAACATCTCTACGT 60
28 AGAACGAGTACGAGACTCCGGCAGATGGCGAACAAACGTAGAGATGTTTTTGGTAACTTT 60
29 CGGAGTCTCGTACTCGTTCTCAGAAAATGCTGGAGGGCACGAGCGGCGAGAACGGTGAGT 60
30 TTACTCACCGTTCTCGCCG 19
PCR:
PCR was run with pgbr22 and stored in -20 degree freezer until Friday